Glutamate is now widely accepted as the major excitatory neurotransmitter in the central nervous system of all vertebrates [see e.g., Nakanishi (1992) Science 258, 597–603]. Abnormalities in glutamatergic synaptic transmission have been implicated in many neuropathologies, including certain seizures, ischemia-induced neuronal death, schizophrenia, Alzheimer's disease, Parkinson's disease, and Huntington's disease [see e.g., Coyle and Puttfarcken (1993) Science 262, 689–695].
Excessive release of glutamate into the synaptic cleft is believed to be a common underlying basis for many of these disease states. There is also evidence that some glutamate receptors such as the NMDA (N-methyl-D-aspartate) and the metabotropic receptors may be involved in neuronal plasticity [see e.g., Bashir et al. (1993) Nature 363, 347–350].
Evidence which has been accumulated for the last decade strongly supports the notion that glutamatergic neurotransmission occurs via an exocytotic process involving the interaction of glutamate-containing synaptic vesicles with the plasma membrane of the presynaptic ending. In support of this is the observation that glutamate is taken up into purified, isolated synaptic vesicles in an ATP-dependent manner [see e.g., Naito and Ueda (1983) J. Biol. Chem. 258, 696–699; and Tabb and Ueda (1991) J. Neurosci. 11, 1822–1828], consistent with immunocytochemical evidence that glutamate is concentrated in synaptic vesicles which are distinct from GABA-vesicles [Storm-Mathisen et al. (1983) Nature 301, 517–520].
Biochemical evidence also suggested that high concentrations of glutamate are accumulated in brain synaptic vesicles in vivo. Studies by Nicholls and co-workers indicate that the exocytotic pool of glutamate originates from a noncytoplasmic site within the nerve terminal [see e.g., Nicholls and Sihra (1986) Nature 321, 772–773; and McMahon and Nicholls (1991) Biochim. Biophys. Acta 1059, 243–264].
Moreover, Kish and Ueda [(1991) Neurosci. Lett. 122, 179–182] provided evidence that vesicular glutamate is released in a calcium-dependent manner from permeabilized synaptosomes. This body of evidence clearly demonstrates that synaptic vesicles are the storage site of the glutamate to be released from nerve terminals.
The vesicular glutamate uptake system has several distinctive properties which distinguish it from the cellular glutamate re-uptake system present in the plasma membrane. It is stringently specific for glutamate, has a relatively high Km, and requires low concentrations of chloride for optimal activity [(Naito and Ueda (1985) J. Neurochem. 44, 99–109; and Fykse et al. (1989) J. Neurochem. 52, 946–951].
The driving force for glutamate uptake is provided by an electrochemical proton gradient generated by a V-type H+-ATPase in the synaptic vesicle membrane [Naito and Ueda (1985) J. Neurochem. 44, 99–109]. The precise mechanism by which the glutamate transporter utilizes this proton gradient to drive glutamate uptake is not fully understood; however, compounds that interfere with the formation of such gradients have a marked inhibitory effect on glutamate transport [Naito and Ueda (1985) J. Neurochem. 44, 99–109; Tabb et al. (1992) J. Biol Chem 267, 15412–15418].
It has been proposed that glutamate uptake into synaptic vesicles represents the critical step in diverting glutamate away from the metabolic pathway and toward the neurotransmitter pathway [Ueda (1986) in Excitatory Amino Acids (Roberts P. J., Storm-Mathisen J., and Bradford H. F., eds), pp. 173–195, Macmillan Press, London]. Thus, it is desirable to regulate the vesicular glutamate uptake system under normal physiological conditions. Alterations in such a regulatory system could cause the abnormalities in glutamatergic neurotransmission implicated in the variety of central nervous system disorders mentioned above. However, while many studies have focused on changes associated with postsynaptic glutamate receptors, few have addressed presynaptic regulation of glutamatergic neurotransmission at the level of vesicular transport. What is needed are compositions and methods for regulating the uptake of glutamate into the synaptic vesicles.